Renin inhibitor and process to make same



OGL-6, 1970 R. R. sMr-:BY ETAL 3,532,724

i BENIN INHIBITOR AND PROCESS T0 MAKE SAME l Filed May 5l, 1967 OOO. OON OO@ NLmH.

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OOmN OOOM m INVENTORS' ROBEFIVR .SMEBY SUBHA VSEN F. MERLIN BUMPUS ATTORNEY J" United States Patent O M U.S. Cl. 260-403 4 Claims ABSTRACT F THE DISCLOSURE There is disclosed herein an inhibitor of renin prepared from mammalian kidney tissue, characterized by possessing the infrared spectrum shown in FIG. 1 and the phospholipid structure in which R1 and R2 represent the hydrocarbon chains of fatty acids containing from 16-20 carbon atoms and from 0-4 double bonds, and R3 represents the hydrocarbon radical of a hydroxyamino acid dilferent from serine, threonine or homoserine containing from 4-10 carbon atoms. The renin inhibitor is useful in the control of blood pressure in hypertension, and methods for its preparation and use are also given.

The invention described herein was made in the course of, or under, a grant from the U.S. Public Health Service, Department of Health, Education, and Welfare.

The present invention relates to a renin inhibitor and to a process for preparing same, as well as to pharmaceutical preparations containing said renin inhibitor. The renin inhibitor of this invention may be represented by the formula:

O GHz-C-ilJ-Rl O CH-O-i'IJ-R2 O CHz-O-g-O-CH-CH-C O OH OH R3 NH2 in which R1 and R2 represent the hdrocarbon radicals of fatty acids containing from 16-20 carbon atoms and from 0-4 double bonds, and R3 represents the hydrocarbon radical of a hydroxyamino acid different from serine, threonine, and homoserine, said radical contaaining from 4-10 carbon atoms.

Renin, an enzyme released from kidney, reacts With an a-Z-globulin in plasma (renin substrate) to release a decapeptide, angiotensin I. Converting enzyme, also present in plasma, rapidly splits histidyl-leucine from the C-terminal end of angiotensin I to yield an octapeptide, angiotensin II. This latter compound is the most potent natural pressor substance known. Both peptides are rapidly degraded by peptidases named angiotensinases. The reninangiotensin system, recently reviewed by Peart in Pharm. Rev. 17, 143 (1965) and Page and Bumpus in Physiol. Rev. 41, 331 (1961) appears to play an important role in the regulation of normal blood pressure, salt metabolism, and some types of experimental and clinical hypertension.

The renin inhibitor of this invention, in inhibiting an essential element of the above system, is useful in the control of blood pressure in hypertension and has the advantage of being ineffective in normotensive subjects. It may be formulated with pharmaceutically acceptable 3,532,724 Patented Oct. 6, 1970 ICC vehicles such as, Ifor example, physiological saline, in the form of suspensions for injection containing from 0.5 to 25 mg./1nl. of the active ingredient. Such preparations for injection may be administered, preferably by intramuscular injection, in doses containing from 0.1-10 mg/ kg. over periods of time of from 2-4 days, when return to normal blood pressure levels will usually be observed. The renin inhibitor of this invention may also be formulated with suitable excipients such as, for example, starch, lactose, magnesium stearate, or magnesium silicate, in the form of tablets or capsules containing from 50-250 mg. of the active ingredient each, and may be administered orally in divided doses as required to control blood pressure levels.

The renin inhibitor of this invention is present in mammalian kidney tissue, and We prefer to use hog kidneys as the starting material for its preparation. More specically, in a preferred procedure, ground hog kidney is extracted with approximately 2.5 parts (W./v.) of acetone, Washed twice with approximately one part of acetone, combining the acetone extracts and Washings and evaporating them under reduced pressure. It should be noted at this point that the renin inhibitor of this invention is present in its natural state in the kidney tissue in a nondialyzeable form, probably bound to protein. Acetone eX- traction obviously breaks the bond between the renin inhibitor of this invention and the protein, because the renin inhibitor is dialyzeable after having been extracted from kidney tissue with acetone.

The acetone-extracted kidney tissue is discarded, and the dry residue from the acetone extraction step 1 part) is dissolved in approximately 7.5 parts of chloroformmethanol mixture 2:1, filtered, the filtrate evaporated to dryness under reduced pressure and redissolved in the same amount of chloroform-methanol mixture 2:1 and then washed five times with 0.2 volume of water each. The aqueous Washings are discarded, and the organic phase is evaporated to dryness under reduced pressure. The residue is dissolved in a minimum of petroleum ether (B.P. 30-60 C.), and ten volumes of acetone are added. The mixture is kept at 20 C. to 30 C., preferably at 24 C., for 24 hours, and is then centrifuged. The supernatant is discarded, and the precipitate is washed three times with acetone, to yield a crude lipid fraction.

The crude lipid fraction obtained above is dissolved in a minimum of chloroform-methanol mixture 4:1 and is chromatographed on a column of silicic acid prepared with the same solvent mixture and containing about 55 parts (W./w.) of silicic acid per part of crude lipid. The chromatogram is developed with about 10 parts (v./W.) of chloroform-methanol mixture 4:1, thus eluting substantially all of the renin inhibitor. Continued elution of the above silicic acid column with successive portions of chloroform-methanol mixtures 3:1, 3:2, and 1:4 yields a lecithin fraction and other phospholipids which are inactive as renin inhibitors.

The eluates obtained above with chloroform-methanol mixtures 4:1 contain not only the desired renin inhibitor, but also phosphatidyl serine, phosphatidyl ethanolamine, and probably also neutral lipids. They are evaporated under nitrogen and under reduced pressure, the resulting lipid fraction is dissolved in a minimum of chloroformmethanol mixture 6:1 and is chromatographed again on a column containing about parts of silicic acid per part of lipid fraction. This second column of silicic acid is eluted rst with chloroform-methanol mixture 6:1 to remove inactive materials, mainly phosphatidyl serine, phosphadityl ethanolamine, and neutral lipids, and then With ethyl acetate-methanol mixture 3:2. This last eluate rontains substantially all of the renin inhibitor of this invention available from the starting material.

If the above renin inhibitor fraction should still contain some phosphatidyl serine it may be purified further by chromatography on a third column of silicic acid prepared in chloroform-methanol 4:1 containing 10 ml. concentrated aqueous ammonia solution per liter. Elution of this last-named silicic acid column with chloroformmethanol mixture 4:1 yields the renin inhibitor of this invention. The process may be represented by the following scheme.

Ground Kidney Extract wilth acetone l Residue Acetone extract discard evaporate to dryness dissolve in CHCla MeOH (2 1) extrast with Witter Water phase Organic phase discard Evaporate to dryness dissolve in pet. ether Add volumes acetone l Supernate Precipitate discard Separate on silicic acid column Lipid applied CHCls: MeOH (3 l) Lecithin Fraction (inactive) l CHCla MeOH 4 1) Cephalin Fraction and Neutral lipids Second Silicic acid column Lipid applied ICHCls: MeOH (6 1) Phosphatidyl ethanolaruine (inactive) l EtOAc MeOH (3 2) Inhibitor other phospholipids neutra1 lipids EXAMPLE l The acetone extract of 3,592 g. of kidney, prepared by extracting with 8.75, 3.5 and 3.5 liters of acetone, is evaporated to dryness under reduced pressure. The residue (135 g.) is extracted with liter of chloroform-methanal mixture (2:1), filtered and evaporated to dryness. It is dissolved in 1 liter of chloroform-methanol (2:1) and the solution is washed 5 times with 1/5 this volume of distilled water at 4. The orgnic phase is then taken to dryness under reduced pressure. The residue (63.7 g.) is dissolved in a minimum of petroleum ether (B.P. 60") and 10 volumes of acetone are added. After 24 hours at -24, the precipitate is removed by centrifugation, and Washed three times with acetone. This crude lipid (18.5 g.) is then fractionated into major phospholipid classes by chromatography.

Silicic acid (75 g.)(Mallinckrodt, 100 mesh, chromatographic grade), activated by heating overnight at 110, is suspended in chloroform-methanol (4:1) system and poured into a column. The ow rate is adjusted 4 to 1.5-2 ml. per minute. After the silicic acid has reached a constant level, the solvent is allowed to flow until it reaches the surface of the silicic` acid forming a packed column of 3 x 40 cm. The phospholipid obtained above (1.35 g.) is dissolved in a small volume of the same chloroform-methanol mixture and pipetted into the column. One gram of silicic acid is used for each 0.8 mg. of phos- I CHCla: MeOH (1 :4)

Other phospholipids (inactive) pholipid phosphorus applied to the column. The column is developed with the following solvents:

( 1) Chloroform:methanol-4:l vol. used 750 ml. (2) Chloroform:methanol3:2 vol. used 350 ml. (3) Chloroform:methanol-1:4 vol. used 350 m1.

The eluate obtained with solvent (l) contains all inhibitor activity and is evaporated to dryness under reduced pressure in a nitrogen atmosphere (yield 0.412 g.). This fraction contained phosphatidyl serine, phosphatidyl ethanolamine, and possibly neutral lipid in addition to the inhibitor and these are separated by chromatography on another silicic acid column.

This column, prepared as described before from 69 g. silicic acid, is 3 x 40 cm. The sample (412 mg.) is applied to the column in chloroformzmethanol (6:1) and is developed with the following solvent systems:

(1) Chloroform:methanol-6:1 vol. used 550 ml. (2) Ethyl acetate:methanol3 :2 vol. used 300 ml'.

The loading factor is 0.5 mg. phospholipid phosphorus for each gram of silicic acid. Eluent from solvent system (2) is pooled and evaporated to dryness in a iilm evaporator under a nitrogen atmosphere (yield 63 mg). If more than 300 ml. of eluting solvent (2) are used, phosphatidyl serine is eluted from the column. If phosphatidyl serine is present in the sample it is removed using a column prepared from 25 g. of silicic acid with chloroform-methanol (4:1) as described above. The finished column (l x 20 cm.) is washed with 30 ml, of chloroform-methanol-ammonia prepared by adding ml. of concentrated ammonium 3. A renin inhibitor characterized by possessing the hydroxide to 1 liter of chloroform-methanol (4:1). The chemical structure sample is applied to the column in chloroform-methanol O (4:1) and the column is developed with the following sol- (HIMO-(Q Rl vent systems: 5 (1) Chloroform:methanol-4:1 vol. used 200 ml. i? o (2) Chloroform:methanol-3:2 vol. used 150 ml. CH-0C-R- (3) Methanol-vol. used 100 ml. 2 The eluate from solvent system (1) contains the inhibitor 10 GHz-o-l-o-CI-I-CII-COOII and when 75 mg. of lipid are applied to the column, 42

0H R3 NH2 mg. are recovered in this fraction.

- in which R1 and R2 are selected from the rou consisting The renin inhibitor obtained 1n the above manner is of hydrocarbon radicals of fatty acids gontaning from characterized by the infrared absorption spectrum of a 16 20 carbon atoms and from 0 4 double bonds and R3 llm offhlniln inhibitor on a Sodlum chlonde crystal l5 is selected from the group consisting of hydrocarbon s own in Furthermore, the renin inhibitor of this invention is .radlcals contammg fr0m.4-10 carbon atoms and which characterized by the following fatty acid composition, s furthmore chtlacteflzed by possessing the followmg determined by gas-liquid chromatography and comparison atty acl composl lons' with known samples: C16 (saturated): 17.7%

C18 (one double bond): 12.4% C20 (four double bonds): 37.9% C18 (saturated): 14.4%

FATTY ACID COMPOSITION oF RESIN INHIBITOR C18 (two double bonds): 6 1% Retention time of- Percent of Unidentified: 11.5% totaitatty Standard Sample acidsin 4. The process for preparing a renin inhibitor com- Chain length: Double bonds (min.) (min.) sample prising:

5.8 17. (a) extracting mammalian kidney tissue with acetone, gj j and evaporating said acetone extract under reduced 15.6 6.1 pressure to dryness; jg (b) redissolving the dry product obtained from said acetone extract in chloroform-methanol 2:1, filtering,

evaporating the filtrate to dryness under reduced pressure, redissolving in chloroform-methanol 2:1, washing with water, and evaporating the organic phase to dryness under reduced pressure;

(c) redissolving said residue from the evaporation of the organic phase in petroleum ether, Vadding ten volumes of acetone, keeping at -20 to 30 C. for 24 hours,

We claim: 1. A renin inhibitor of the formula O 40 centrifuging, and washing the precipitate with acetone; CH2-O G-R (d) redissolving said precipitate in chloroform-methanol o 4:1, chromatographing on silicic acid, eluting with CH O R2 chloroform-methanol 4:1, and evaporating the eluates O to dryness under nitrogen and under reduced pressure; olz-o-g-o-oH-oiI-ooon (e) redissolving the residue obtained in the preceding H R3 NH2 step in chloroform-methanol 6: 1, and chromatographing on silicic acid, eluting iirst with chloroformmethanol 6:1 to remove inactive material and then eluting with ethyl acetate-methanol 3:2, to obtain the in which R1 and R2 are Selected from the group consisting renin inhibitor by evaporation under reduced presof hydrocarbon radicals of fatty acids containing from Sure 0f Sald ethyl atate'methanol eluates- 16-20 carbon atoms and from 0-4 double bonds, and 5. R3 is selected from the group consisting of hydrocarbon o References Cmd radicals containing from 4-10 carbon atoms. UNITED STATES PATENTS 2. A renin inhibitor characterized by the infrared ab- 3,197,371 7/ 1965 Dailey et al 167-65 sorption spectrum of a film thereof on a sodium chloride crystal shown in FIG. 1. ELBERT L. ROBERTS, Primary Examiner 

